The overall objective of this project is to isolate and to characterize the proteins involved in the two types of serotonin receptors. This study will be conducted using two new tools developed in our laboratory: a serotonin photoaffinity labeling probe and a serotonin affinity column. The information which this study will provide is essential for understanding the molecular mechanism of serotonin neuronal function. We will establish the specificity of our serotonin photoaffinity labeling probe-nitroarylazidophenyl-serotonin (NAP-Serotonin). Three distinct polypeptides have been labeled with this photoaffinity probe and have been separated by SDS-polyacrylamide gel electrophoresis. The role of these three proteins in 5-HT neurotransmission will be determined. The molecular nature of the two types of 5-HT receptors will be investigated for the first time. The topology of the two types of serotonin receptors within the neuronal membrane will be examined by studying the effect of the membrane fluidity upon the binding activities. Furthermore, the two types of active serotonin receptors will be solubilized and purified to homogeneity by a combination of affinity, gel filtration and ion exchange column chromatography. The binding properties of these purified serotonin-binding-proteins will be systematically studied using a radioligand binding assay. The interaction of antidepressants such as amitriptyline and mianserin with the purified serotonin binding proteins will be studied. In addition, the serotonin-binding-proteins will be evaluated for adenylate cyclase activity. By defining the molecular properties of the serotonin receptors we will help to elucidate the mechanism of action of certain drugs presently used to treat mental disorders as well as suggesting new uses of drugs.